Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission

Significance The results reported here uncover a uniquely evolved aquaporin in malaria parasites, which is important for production of sporozoites during the mosquito phase of the parasite lifecycle. Sporozoites are the parasite form responsible for malaria transmission to humans following a mosquito bite. Aquaporins are known transporters of water and other molecules across biological membranes. The aquaporin has unusual structural features and inhabits a vesicle-like organelle inside parasite cells. Since aquaporins can be targeted by drugs, we propose that this aquaporin may be a promising target of future antimalarial interventions.


Fig. S2 .
Fig. S2.Predicted membrane topology of PfAQP2 (left) and PbAQP2 (right).Topology of is predicted with Alphafold and PPM, and visualized using Protter.Transmembrane helices are labeled as H1-6, half-helices as HH1-2, and extramembrane loops as LA-E.Key residues are highlighted: selectivity filter in red, NPA/NPM motifs in black, FST triad of loop D in green, and histidine participating in the pore in cyan.

Fig. S4 .
Fig. S4.Putative 3D structures of PfAQP2 and PbAQP2.Protein structures are predicted by Alphafold Monomer v2.0 and can be accessed in UniProt, PlasmoDB and other databases as C0H4T7 and A0A509AUY5, respectively.Structures are colored by Alphafold prediction confidence values as shown in the key below the structures.

Fig. S5 .
Fig. S5.Predicted structure of PbAQP2 pore and selectivity filter.Structures are predicted with Alphafold Monomer v2.0 on the CoLab server.Top ranking models are shown.Structures are colored by confidence level as shown in the key below the figure.Residues contributing to the selectivity filter are shown in red and those contributing to the NPA/NPM motif are shown in cyan, while the histidine residue participating in the pore is shown in magenta.

Fig. S6 .
Fig. S6.Schematic diagram of Δpfaqp2 knockout generation by CRISPR-Cas9.(A) The endogenous NF54 PfAQP2 locus shown at the top was disrupted using the AQP2-dis-pDC2 plasmid construct shown in the center of the panel resulting in the disrupted locus shown at the bottom.For the generation of the plasmid construct, primers described in TableS5are used to clone the 5¢ and 3¢ donor homology arms (HA) upstream and downstream of the Cas9 cut site on the parasite genome, respectively.The donor AQP2-dis-pDC2 plasmid includes the hDHFR selectable marker cassette.(B) PCR genotypic analysis of successfully generated recombinants (left picture) and isolated Δpfaqp2 clonal populations (right picture) using primers specific for recombinant genomic sequence.

Fig. S7 .
Fig. S7.Generation of Δpbaqp2 mutant and gene tagged parasites.(A) Schematic representation of the disruption of P. berghei in the c507 line, using PlasmoGEM vectors carrying the human DHFR (hDHFR)/yFCU pyrimethamine resistance cassette.In each panel, the wt genomic locus (top), the transfected DNA fragment carrying the homologous arms (HA) and the selectable markers (middle), and the final transgenic locus after gene disruption by double crossover homologous recombination (bottom) are shown.Constructs are not drawn to scale.Small arrows show the primers used for the confirmation of integration of gene targeting constructs and deletion of the gene of interest using PCR as shown in the panels on the right.The c507 wt parental parasites were used as a control for detection of the wt locus.(B) Schematic representation of the 3XHA tagging of in the c507 line by using PlasmoGEM gene tagging vectors that carry the hDHFR)/yFCU selectable marker.In each panel, the wt genomic locus (top), the plasmid carrying the homologous arms (HA), the transfected DNA fragment carrying the tagging cassettes and the selectable markers (middle), and the final transgenic locus after double crossover homologous recombination (bottom) are shown.Constructs are not drawn to scale.Small arrows show the primers used for the confirmation of integration of gene targeting constructs and deletion of the gene of interest using diagnostic PCR reactions as shown in the panels on the right.

Fig. S8 .
Fig. S8.Sporozoite production in Δpbaqp2 mutant oocysts.Midgut sporozoite load in Δpbaqp2 and c507 (control) infected A. coluzzii mosquitoes at 15, 16, 18 and 20 days pbf.The mean of the pooled data from two biological replicates is shown.The number of sporozoites was from suspensions of 20 homogenized midguts, assayed in two batches of ten.Error bars show standard error.

Table S1 .
Oocyst counts of control Δpfaqp2 mutant parasites and NF54 controls in A. coluzzii Infection data from four biological replicates of A. coluzzii infected with Δpfaqp2 (clones G4 and H6) and NF54 parasite lines, assessed at 9 days post-infection.P values were calculated using the Kruskal-Wallis test, comparing to wild type, and adjusted with Dunn's multiple comparisons test.

Table S2 .
Sporozoite numbers of control NF54 and Δpfaqp2 parasites in A. coluzzii infections Mean oocyst and salivary gland sporozoite numbers of A. coluzzii infected with Δpfaqp2 (clones G4 and H6) and NF54 parasite lines.Pooled data from 4 biological replicates are presented.In each replicate, sporozoite numbers were determined from 25-30 homogenized mosquito midguts or salivary glands at 11 and 18 days pbf, respectively.P values were calculated by one-way ANOVA, comparing to wild type, and adjusted with Dunnett's multiple comparisons test.SEM represents standard error of mean.

Table S3 .
Oocyst numbers of control c507 and Δpbaqp2 parasites in A. coluzzii infections Data are from two biological replicates of infected A. coluzzii dissected at 8 days pbf.P values were calculated using the Mann-Whitney t-test.

Table S4 .
Sporozoite numbers of control c507 and Δpbaqp2 parasites in A. coluzzii infections Mean oocyst and salivary gland sporozoite numbers from A. coluzzii infections.Pooled data from two biological replicates are presented.In each replicate, sporozoite numbers were determined from 25-30 homogenized mosquito midguts or salivary glands at 15 and 21 days pbf, respectively.P values were calculated using the unpaired Student's t-test.SEM represents standard error of mean.

Table S5 .
Oligonucleotide primers used in this studyOverlap sequences for Gibson assembly and restriction sites are shown as underlined.F, forward; R, reverse; WT, wild-type; KO, knockout.All primers are listed in a 5¢ to 3¢ direction.